Oligomer barcode for labeling extracellular vesicles

Abstract

This application relates to oligomer barcodes for labeling extracellular vesicles (EVs). The labeled extracellular vesicles can be used to monitor EV movement for research purposes and for the development of treatments in a variety of diseases and disorders.

1 . A method, comprising:
 obtaining an extracellular vesicle;   attaching a first linker to the extracellular vesicle;   incubating the extracellular vesicle with an oligonucleotide label comprising a second linker, thereby conjugating the oligonucleotide label to the extracellular vesicle;   applying the conjugated extracellular vesicle to a subject or to a sample derived from the subject; and   detecting the presence of the conjugated extracellular vesicle in at least one cell of the subject.   
     
     
         2 . The method of  claim 1 , wherein the first linker comprises 4-formylbenzoate (4FB) and the second linker comprises 6-hydrazinonicotinate acetone hydrazone (HyNic). 
     
     
         3 . The method of  claim 1 , wherein the oligonucleotide label comprises:
 a primary amine conjugated to the second linker, and/or   at least one phosphorothioate bond.   
     
     
         4 . The method of  claim 1 , wherein a length of the oligonucleotide label is in a range of about 10 to about 80 nucleotides. 
     
     
         5 . The method of  claim 1 , wherein the extracellular vesicle and the oligonucleotide label are incubated at a ratio in a range of about 1:10 to about 1:100. 
     
     
         6 . The method of  claim 1 , wherein the extracellular vesicle and the oligonucleotide label are incubated at a ratio of about 1:80. 
     
     
         7 . The method of  claim 1 , wherein the detecting comprises:
 extracting the at least one cell from the subject; and/or   performing polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), DNA scope, RNA scope, isothermal amplification, or unbiased DNA sequencing.   
     
     
         8 . An extracellular vesicle comprising an oligonucleotide label, the oligonucleotide label comprising at least one phosphorothioate bond. 
     
     
         9 . The extracellular vesicle of  claim 8 , wherein the extracellular vesicle is derived from a living subject or in vitro cells. 
     
     
         10 . The extracellular vesicle of  claim 8 , wherein a length of oligonucleotide label is in a range of about 10 to about 80 nucleotides, and/or
 wherein the oligonucleotide label comprises a primary amine.   
     
     
         11 . A method, comprising:
 administering a solution comprising the extracellular vesicle of  claim 8  to a living subject or a sample derived from the living subject; and   detecting the extracellular vesicle in at least one cell of the living subject.   
     
     
         12 . The method of  claim 11 , wherein the living subject is a mouse, a rat, a mammal, a non-human primate, or human. 
     
     
         13 . The method of  claim 11 , wherein the detecting comprises at least one of:
 extracting the at least one cell from the living subject;   lysing the at least one cell of the living subject; or   performing polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), DNA scope, RNA scope, isothermal amplification, or unbiased DNA sequencing.   
     
     
         14 . The method of  claim 11 , wherein the detecting comprises examining the sample under a microscope, the method further comprising:
 fixing the sample onto a slide.   
     
     
         15 . The method of  claim 11 , wherein the sample comprises at least two cell types, the method further comprising:
 detecting the extracellular vesicle in each of the at least two cell types.   
     
     
         16 . A method, comprising:
 analyzing PCR data from a sample; and   based on analyzing the PCR data, determining whether the extracellular vesicle of  claim 8  is present in the sample.   
     
     
         17 . A kit, comprising:
 an oligonucleotide barcode comprising a first linker;   a second linker; and   reagents configured to conjugate the second linker to an extracellular vesicle.   
     
     
         18 . The kit of  claim 17 , wherein the first linker comprises 4-formylbenzoate (4FB) and the second linker comprises 6-hydrazinonicotinate acetone hydrazone (HyNic). 
     
     
         19 . The kit of  claim 17 , wherein the reagents are for covalent conjugation, lipofection, or electroporation. 
     
     
         20 . The kit of  claim 17 , further comprising:
 reagents for purification of extracellular vesicles from a tissue sample; and/or   polymerase chain reaction (PCR) reagents for detection of the oligonucleotide barcode.